Screening assays for identifying differentiation-inducing agents and production of differentiated cells for cell therapy

Abstract

The invention relates to assays for screening growth factors, adhesion molecules, immunostimulatory molecules, extracellular matrix components and other materials, alone or in combination, simultaneously or temporally, for the ability to induce directed differentiation of pluripotent and multipotent stem cells.

What is claimed:  
     
         1 . A method for evaluating the differentiation of totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, in response to one or more chemical or biological agents or physical conditions, comprising: 
 (a) separating individual totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, or groups of such cells, in culture medium into one or a plurality of separate wells which may be open or closed, which wells may be in the same or different apparatus;    (b) exposing said separate wells of cells to one or more putative differentiation-inducing conditions simultaneously or sequentially; and    (c) screening said individual cells or groups of cells to detect markers of differentiation of said individual cells or groups of cells.    
     
     
         2 . The method of  claim 1 , wherein said totipotent, nearly totipotent, or pluripotent stem cells are selected from the group consisting of inner cell mass (ICM) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryos consisting of one to about 400 cells, embryoid body cells, morula-derived cells, embryonic pluripotent cells, and cells therefrom.  
     
     
         3 . The method of  claim 1 , wherein said nearly totipotent, or pluripotent stem cells, or cells therefrom, are selected from the group consisting of human cells, primate cells, bovine cells, porcine cells, murine cells, rat cells, sheep cells, canine and feline cells.  
     
     
         4 . The method of  claim 1 , wherein said one or more putative differentiation-inducing conditions are selected from the group consisting of growth factors, cytokines, tissue extracts, nucleic acids, factors involved in cell-to-cell interactions, adhesion molecules and extracellular matrix components, extracts of extracellular components from tissue, media components, environmental conditions, and living cells that induce differentiation by cell-cell interactions.  
     
     
         5 . The method of  claim 4 , wherein said growth factors, chemokines, and cytokines are selected from the group consisting of the Fibroblast Growth Factor family of proteins (FGF1-23) including but not limited to FGF basic (146 aa), FGF basic (157 AA), FGF acidic, the TGF beta family of proteins including but not limited to TGF-beta 1, TGF-beta 2, TGF-beta sRII, Latent TGF-beta, the Tumor necrosis factor (TNF) superfamily (TNFSF) including but not limited to TNFSF1-18, including TNF-alpha, TNF-beta, the insulin-like growth factor family incuding but not limited to IGF-1 and their binding proteins including but not limited to IGFBP-1, II-1 R rp2, IGFBP-5, IGFBP-6, the matrix metalloproteinases including but not limited to MMP-1, CF, MMP-2, CF, MMP-2 (NSA-expressed), CF, MMP-7, MMp-8, MMP-10, MMP-9, TIMP-1, CF, TIMP-2 and other growth factors and cytokines including but not limited to PDGF, Flt-3 ligand, Fas Ligand, B7-1 (CD80), B7-2(CD86), DR6, IL-13 R alpha, IL-15 R alpha, GRO beta/CXCL2 (aa 39-107), IL 1-18, II-8/CXCL8, GDNF, G-CSF, GM-CSF, M-GSF, PDGF-BB, PDGF-AA, PDGF-AB, IL-2 sR alpha, IL-2 sR beta, Soluble TNF RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR, beta-ECGF, TGF-alpha, TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF, LIF sR alpha, LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF, CNTF, Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF, Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, I-309/CCL1, GRO alpha/CXCL1, GRO beta/CXCL2, GRO gamma/CXCL3, Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, IFN-gamma, Erythropoietin, Thrombopoietin, MIF, IGF-I, IGF-II, VEGF, HGF, Oncostatin M, HRG-alpha (EGF Domain), TGF-beta 2, CNTF R alpha, Tie-2/Fc Chimera, BMP-4, BMPR-IA, Eotaxin/CCL11, VEGF R1 (Fit-1), PDGF sR alpha, HCC-1/CCL14, CTLA-4, MCP-4/CCL13, GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A, Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94), TRAIL R1 (DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP, BMP-2, HVEM/VEGF R2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3 beta/CCL19, Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17, 6Ckine/CCL21, p75 Neurotrophin R (NGF R), SMDF, Neurturin, Leptin R/Fc Chimera, MIG/CXCL9, NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1 (CT-1), GFR alpha-2, BMP-5, IL-8/CXCL8 (Endothelial Cell Derived), Tie-1, Viral CMV UL146, VEGF-D, Angiopoietin-2, Inhibin A, TRANCE/RANK L, CD6/Fc Chimera, CF, dMIP-1 delta/LKN-1/CCL15(68 aa), TRAIL R3/Fc Chimera, Soluble TNF RI, Activin RIA, EphA1, E-Cadherin, ENA-70, ENA-74, Eotaxin-3/CCL26, ALCAM, FGFR1 alpha (IIIc), Activin B, FGFT1 beta (IIIc), LIGHT, FGFR2 beta(IIIb), DNAM-1, Follistatin, GFR alpha-3, gp 130, I-TAC/CXCL11, IFN-gamma R1, IGFBP-2, IGFBP-3, Inhibin B, Prolactin CF, RANK, FGFR2 beta (IIIc), FGFR4, TrkB, GITR, MSP R, GITR Ligand, Lymphotactin/XCL1, FGFR2 alpha (IIIc), Activin AB, ICAM-3 (CD50), ICAM-1 (CD54), TNF RII, L-Selectin (CD62L, BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2 (CD102), IGFBP-4, Osteoprotegerin)OPG), uPAR, Activin RIB, VCAM-1 (CD106), CF, BMPR-II, IL-18 R, IL-12 R beta 1, Dtk, LBP, SDF-1 alpha (PBSF)/CXCL12 (synthetic), E-Selectin (CD62E), L-Selectin (CD62L), P-Selectin (CD62P), ICAM-1 (CD54), VCAM-1 (CD106), CD31 (PECAM-1), hedgehog family of proteins, Interleukin-10, Epidermal Growth Factor, Heregulin, HER4, Heparin Binding Epidermal Growth Factor, bFGF, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B, leptin, Interferon A, Interferon A/D, Interferon B, Interferon Inducible Protein-10, Insulin Like Growth Factor-II, IGBFBP/IGF-1 Complex, C10, Cytokine Induced Neutrophil Chemoattractant 2, Cytokine Induced Neutrophil Chemoattractant 2B, Cytokine Induced Neutrophil Chemoattractant 1, Cytokine Responsive Gene-2, and any fragment thereof and their neutralizing antibodies.  
     
     
         6 . The method of  claim 4 , wherein said factors involved in cell-cell interactions are selected from the group consisting of the ADAM (A Disintegrin and Metalloproteinase) family of proteins including ADAM 1, 2, 3A, 3B, 4-31 and TS1-9, ADAMTSs (ADAMs with thrombospondin motifs), Reprolysins, metzincins, zincins, and zinc metalloproteinases and their neutralizing antibodies.  
     
     
         7 . The method of  claim 4 , wherein said adhesion molecules are selected from the group consisting of Ig superfamily CAM's, Integrins, Cadherins and Selectins and their neutralizing antibodies.  
     
     
         8 . The method of  claim 4 , wherein said nucleic acids that may be tested include but are not limited to those that encode or block by antisense, ribozyme activity, or RNA interference with transcription factors that are involved in regulating gene expression during differentiation, genes for growth factors, cytokines, and extracellular matrix components, or other molecular activities that regulate differentiation.  
     
     
         9 . The method of  claim 4  wherein said cell-cell interactions include placing the cells being assayed in cell-cell contact with cells of another differentiated cell type, or in the presence of media conditioned by cells of another differentiated cell type.  
     
     
         10 . The method of  claim 4  wherein said tissue extracts include but are not limited to materials derived from early stage embryos, fetuses, or adult tissues that may be tested include but are not limited to acellular extracellular matrix prepared by the detergent extraction of tissue from embryoid bodies, primitive endoderm, mesoderm, and ectoderm, and the anlagen of differentiating organs and tissues or living cells or tissues that when co-cultured with the subject cells cause an induction of differentiation.  
     
     
         11 . The method of  claim 4  wherein said environmental conditions include but are not limited to oxygen tension, carbon dioxide tension, nitric oxide tension, temperature, pH, mechanical stress, altered culture substrates such as two vs. three dimensional substrates, growth on beads, inside cylinders, or porous substrates.  
     
     
         12 . The method of  claim 4 , wherein said extracellular matrix components are selected from the group including but are not limited to Keratin Sulphate Proteoglycan, Laminin, Chondroitin Sulphate A, SPARC, beta amyloid precursor protein, beta amyloid, presenilin 1,2, apolipoprotein E, thrombospondin-1,2, Heparan Sulphate, Heparan sulphate proteoglycan, Matrigel, Aggregan, Biglycan, Poly-L-Ornithine, the collagen family including but not limited to Collagen I-IV, Poly-D-Lysine, Ecistatin (Viper Venom), Flavoridin (Viper Venom), Kistrin (Viper Venom), Vitronectin, Superfibronectin, Fibronectin Adhesion-Promoting peptide, Fibronectin Fragment III-C, Fibronectin Fragment-30 KDA, Fibronectin-Like Polymer, Fibronectin Fragment 45 KDA, Fibronectin Fragment 70 KDA, Asialoganglioside-GM, Disialoganglioside-GOLA, Monosialo Ganglioside-GM 1 , Monosialoganglioside-GM 2 , Monosialoganglioside-GM 3, , Methylcellulose, Keratin Sulphate Proteoglycam, Laminin and Chondroitin Sulphate A.  
     
     
         13 . The method of  claim 1 , wherein said individual cells or individual groups of cells are separated into separate wells of one or more multi-well plates.  
     
     
         14 . The method of  claim 1 , further comprising: 
 isolating primary and/or progenitor cells from reference tissues and placing said primary and/or progenitor cells into separate vessels of a microarray thereby forming a control reference library; and    after exposing said separate vessels of totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, to said one or more putative differentiation-inducing compounds either simultaneously or sequentially;    comparing said individual totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, or groups of cells, to said reference library in order to evaluate the differentiation of said individual cells or groups of cells.    
     
     
         15 . The method of  claim 14 , wherein said reference library of primary cells is constructed in one or more multi-well plates.  
     
     
         16 . The method of  claim 14 , wherein said primary and/or progenitor cells for said reference library include one or more of brain cells, heart cells, liver cells, skin cells, pancreatic cells, blood cells, reproductive cells, nerve cells, sensory cells, vascular cells, skeletal cells, immune cells, lung cells, muscle cells, and kidney cells.  
     
     
         17 . The method of  claim 14 , wherein said totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, and said reference library cells are compared using functional assays specific for the particular primary cells in the reference library.  
     
     
         18 . The method of  claim 17 , wherein said functional assays measure the production of enzymes or metabolites produced by said reference primary and/or progenitor cells.  
     
     
         19 . The method of  claim 14 , wherein said exposed totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, and said reference library cells are compared by testing for the presence of specific cell surface markers or receptors.  
     
     
         20 . The method of  claim 19 , wherein the presence of said cell surface markers is detected using labeled antibodies or ligands specific for said cell surface markers or receptors.  
     
     
         21 . A method for evaluating the differentiation of a totipotent, nearly totipotent, or pluripotent stem cell, or cells therefrom, or a group of such cells, in response to different compounds or combinations of compounds, comprising: 
 (a) separating individual totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, or groups of such cells, into one or a plurality of separate vessels which may be open or closed, which vessels may be in the same or different apparatus;    (b) exposing said separate vessels of cells to a panel of different putative differentiation-inducing compounds or combinations thereof simultaneously or sequentially; and    (c) comparing said individual cells or groups of cells to a reference panel of differentiated or partially differentiated cells in order to evaluate the differentiation of said individual totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, or groups of such cells.    
     
     
         22 . The method of  claim 21 , wherein said totipotent, nearly totipotent, or pluripotent stem cells, are selected from the group consisting of inner cell mass (ICM) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryos consisting of one to about 400 cells, embryoid body cells, morula-derived cells, embryonic pluripotent cells, and cells therefrom.  
     
     
         23 . The method of  claim 21 , wherein said individual totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, are selected from the group consisting of human cells, primate cells, bovine cells, porcine cells, murine cells, rat cells, sheep cells, and rabbit cells.  
     
     
         24 . The method of  claim 21 , wherein said one or more putative differentiation-inducing compounds are selected from the group consisting of growth factors, cytokines, factors involved in cell-to-cell interactions, adhesion molecules and extracellular matrix components.  
     
     
         25 . The method of  claim 24 , wherein said growth factors and cytokines are selected from the group consisting of TGF-beta 2, PDGF, Fas Ligand, FGF acidic, B7-1 (CD80), B7-2(CD86), DR6, IL-13 R alpha, IL-15 R alpha, GRO beta/CXCL2 (aa 39-107), IL 1-18, II-8/CXCL8, TNF-alpha, TNF-beta, GDNF, G-CSF, GM-CSF, M-GSF, PDGF-BB, PDGF-M, PDGF-AB, IL-2 sR alpha, IL-2 sR beta, Soluble TNF RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR, beta-ECGF, FGF basic (146 aa), FGF basic (157 AA), FGF1-21, TGF-alpha, TGF-beta 1, TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF, LIF sR alpha, LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF, CNTF, Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF, Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, I-309/CCL1, GRO alpha/CXCL1, GRO beta/CXCL2, GRO gamma/CXCL3, Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, IFN-gamma, Erythropoietin, Thrombopoietin, MIF, IGF-I, IGF-II, VEGF, HGF, Oncostatin M, HRG-alpha (EGF Domain), Latent TGF-beta, TGF-beta 2, CNTF R alpha, Tie-2/Fc Chimera, BMP-4, BMPR-IA, Eotaxin/CCL11, VEGF R1 (Fit-1), PDGF sR alpha, HCC-1/CCL14, CTLA-4, MCP-4/CCL13, GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A, TGF-beta sRII, Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94), TRAIL R1 (DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP, BMP-2, HVEM/VEGF R2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3 beta/CCL19, Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17, 6Ckine/CCL21, p75 Neurotrophin R (NGF R), SMDF, Neurturin, Leptin R/Fc Chimera, MIG/CXCL9, NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1 (CT-1), GFR alpha-2, BMP-5, IL-8/CXCL8 (Endothelial Cell Derived), Tie-1, Viral CMV UL146, VEGF-D, Angiopoietin-2, Inhibin A, TRANCE/RANK L, CD6/Fc Chimera, CF, dMIP-1 delta/LKN-1/CCL15(68 aa), TRAIL R3/Fc Chimera, Soluble TNF RI, Activin RIA, EphA1, E-Cadherin, ENA-70, ENA-74, Eotaxin-3/CCL26, ALCAM, FGFR1 alpha (IIIc), Activin B, FGFT1 beta (IIIc), LIGHT, FGFR2 beta(IIIb), DNAM-1, Follistatin, GFR alpha-3, gp 130, I-TAC/CXCL11, IFN-gamma R1, IGFBP-2, IGFBP-3, Inhibin B, Prolactin CF, RANK, FGFR2 beta (IIIc), FGFR4, TrkB, GITR, MSP R, GITR Ligand, Lymphotactin/XCL1, FGFR2 alpha (IIIc), Activin AB, ICAM-3 (CD50), ICAM-1 (CD54), TNF RII, L-Selectin (CD62L, BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2 (CD102), IGFBP-4, Osteoprotegerin)OPG), uPAR, Activin RIB, VCAM-1 (CD106), CF, BMPR-II, IL-18 R, IL-12 R beta 1, Dtk, LBP, IGFBP-1, II-1 R rp2, IGFBP-5, IGFBP-6, MMP-1, CF, MMP-2, CF, MMP-2 (NSA-expressed), CF, MMP-7, MMp-8, MMP-10, CF, MMP-9<CF, TIMP-1, CF, TIMP-2, CF, SDF-1 alpha (PBSF)/CXCL12 (synthetic), E-Selectin (CD62E), L-Selectin (CD62L), P-Selectin (CD62P), ICAM-1 (CD54), VCAM-1 (CD106), CD31 (PECAM-1).  
     
     
         26 . The method of  claim 24 , wherein said factors involved in cell-cell interactions are selected from the group consisting of ADAM 1, 2, 3A, 3B, 4-31, TS1-9.  
     
     
         27 . The method of  claim 24 , wherein said adhesion molecules are selected from the group consisting of Ig superfamily CAM's, Integrins, Cadherins and Selectins.  
     
     
         28 . The method of  claim 24 , wherein said extracellular matrix components are selected from the group consisting of Keratin Sulphate Proteoglycan, Laminin, Chondroitin Sulphate A, Thrombospondin-1, Heparan Sulphate, Aggregan, Biglycan, Poly-L-Ornithine, Collagen I, Collagen II, Collagen IV, Poly-D-Lysine, Ecistatin (Viper Venom), Flavoridin (Viper Venom), Kistrin (Viper Venom), Vitronectin, Superfibronectin, Fibronectin Adhesion-Promoting peptide, Fibronectin Fragment Ill-C, Fibronectin Fragment-30 KDA, Fibronectin-Like Polymer, Fibronectin Fragment 45 KDA, Fibronectin Fragment 70 KDA, Prostaglandin F 2 , Somatostatin, Thyrotropin Releasing Hormone, L-Thyroxine, 3,3,5-Triiodo-L-Thyronine, L-Ascorbic Acid, Asialoganglioside-GM, Disialoganglioside-GOLA, Monosialo Ganglioside-GM 1 , Monosialoganglioside-GM 2 , Monosialoganglioside-GM 3 , Lipids, Transferrin, B-Cyclodextrin, Ascorbate, Fetuin, Heparin, 2-Mercaptoethanol, Horse Serum, DMSO, Chicken Serum, Goat Serum, Rabbit Serum, Human Serum, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B, Pituitary Extract, Stromal Cell Factor, Conditioned Medium, Hybridoma Medium, d-Aldosterone, Dexamethasone, DHT, B-Estradiol, Glucagon, Insulin, Progesterone, Prostaglandin-D 2 , Prostaglandin-E 1 , Prostaglandin-E 2 , Prostaglandin-F 2 , Serum-Free Medium, Gene Therapy Medium, MDBK-GM Medium, QBSF-S1, Endothelial Medium, Keratinocyte Medium, Melanocyte Medium, Interleukin-10, Epidermal Growth Factor, Heparin Binding Epidermal Growth Factor, bFGF, Gly-His-Lys, Insulin Like Growth Factor-II, IGBFBP/IGF-1 Complex, C10, Cytokine Induced Neutrophil Chemoattractant 2, Cytokine Induced Neutrophil Chemoattractant 2B, Cytokine Induced Neutrophil Chemoattractant 1, Cytokine Responsive Gene-2, Endothelial Cell Growth Supplement, Interferon A, Interferon A/D, Interferon B, Interferon Inducible Protein-10, Leptin, Methylcellulose, Keratin Sulphate Proteoglycam, Laminin and Chondroitin Sulphate A.  
     
     
         29 . The method of  claim 21 , wherein said individual totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, or individual groups of such cells are separated into separate wells of one or more multi-well plates.  
     
     
         30 . The method of  claim 21 , wherein said reference differentiated or partially differentiated cell is a primary or progenitor cell selected from the group consisting of brain cells, heart cells, liver cells, skin cells, pancreatic cells, blood cells, reproductive cells, nerve cells, sensory cells, vascular cells, skeletal cells, immune cells, lung cells, muscle cells, and kidney cells.  
     
     
         31 . The method of  claim 21 , wherein said exposed totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, and said reference cell are compared using functional assays specific for the reference cell.  
     
     
         32 . The method of  claim 31 , wherein said functional assay measures the production of one or more enzymes or metabolites produced by said reference cell.  
     
     
         33 . The method of  claim 21 , wherein said exposed totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, and said reference cell are compared by testing for the presence of specific cell surface markers or receptors.  
     
     
         34 . The method of  claim 33 , wherein the presence of said cell surface markers is detected using labeled antibodies or ligands specific for said cell surface markers or receptors.  
     
     
         35 . The method of  claim 21 , wherein said exposed totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, and said reference cell are compared by comparing RNA expression profiles.  
     
     
         36 . A method for evaluating the differentiation of a totipotent, nearly totipotent, or pluripotent stem cell, or cells therefrom, or a group of such cells, in response to different compounds or combinations of compounds, comprising: 
 (a) isolating a transfected totipotent, nearly totipotent, or pluripotent stem cell, or cells therefrom, wherein said cell is transfected with at least one reporter gene, the expression of which is operably linked to a promoter that is activated when the cell is induced to differentiate or partially differentiate;    (b) expanding said transfected cell in culture;    (c) separating individual transfected cells or individual groups of cells into one or a plurality of separate vessels which may be open or closed, which vessels may be in the same or different apparatus;    (d) systematically exposing said separate vessels of transfected cells simultaneously or sequentially to a panel of different putative differentiation-inducing compounds or combinations thereof; and    (e) analyzing said individual transfected cells or groups of cells in order to detect expression of said at least one reporter gene.    
     
     
         37 . The method of  claim 35 , wherein said totipotent, nearly totipotent, or pluripotent stem cells, are selected from the group consisting of inner cell mass (ICM) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryos consisting of one to about 400 cells, embryoid body cells, morula-derived cells, embryonic pluripotent cells, and cells therefrom.  
     
     
         38 . The method of  claim 35 , wherein said totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, are selected from the group consisting of human cells, primate cells, bovine cells, porcine cells, murine cells, rat cells and sheep cells.  
     
     
         39 . The method of  claim 35 , wherein said one or more putative differentiation-inducing compounds are selected from the group consisting of growth factors, cytokines, factors involved in cell-to-cell interactions, adhesion molecules and extracellular matrix components.  
     
     
         40 . The method of  claim 39 , wherein said growth factors and cytokines are selected from the group consisting of TGF-beta 2, PDGF, Fas Ligand, FGF acidic, B7-1(CD80), B7-2(CD86), DR6, IL-13 R alpha, IL-15 R alpha, GRO beta/CXCL2 (aa 39-107), IL 1-18, II-8/CXCL8, TNF-alpha, TNF-beta, GDNF, G-CSF, GM-CSF, M-GSF, PDGF-BB, PDGF-AA, PDGF-AB, IL-2 sR alpha, IL-2 sR beta, Soluble TNF RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR, beta-ECGF, FGF basic (146 aa), FGF basic (157 M), FGF1-21, TGF-alpha, TGF-beta 1, TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF, LIF sR alpha, LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF, CNTF, Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF, Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, I-309/CCL1, GRO alpha/CXCL1, GRO beta/CXCL2, GRO gamma/CXCL3, Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, IFN-gamma, Erythropoietin, Thrombopoietin, MIF, IGF-I, IGF-II, VEGF, HGF, Oncostatin M, HRG-alpha (EGF Domain), Latent TGF-beta, TGF-beta 2, CNTF R alpha, Tie-2/Fc Chimera, BMP-4, BMPR-IA, Eotaxin/CCL11, VEGF R1 (Fit-1), PDGF sR alpha, HCC-1/CCL14, CTLA-4, MCP-4/CCL13, GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A, TGF-beta sRII, Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94), TRAIL R1 (DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP, BMP-2, HVEM/VEGF R2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3 beta/CCL19, Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17, 6Ckine/CCL21, p75 Neurotrophin R (NGF R), SMDF, Neurturin, Leptin R/Fc Chimera, MIG/CXCL9, NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1 (CT-1), GFR alpha-2, BMP-5, IL-8/CXCL8 (Endothelial Cell Derived), Tie-1, Viral CMV UL146, VEGF-D, Angiopoietin-2, Inhibin A, TRANCE/RANK L, CD6/Fc Chimera, CF, dMIP-1 delta/LKN-1/CCL15(68 aa), TRAIL R3/Fc Chimera, Soluble TNF RI, Activin RIA, EphA1, E-Cadherin, ENA-70, ENA-74, Eotaxin-3/CCL26, ALCAM, FGFR1 alpha (IIIc), Activin B, FGFT1 beta (IIIc), LIGHT, FGFR2 beta(IIIb), DNAM-1, Follistatin, GFR alpha-3, gp 130, I-TAC/CXCL11, IFN-gamma RI, IGFBP-2, IGFBP-3, Inhibin B, Prolactin CF, RANK, FGFR2 beta (IIIc), FGFR4, TrkB, GITR, MSP R, GITR Ligand, Lymphotactin/XCL1, FGFR2 alpha (IIIc), Activin AB, ICAM-3 (CD50), ICAM-1 (CD54), TNF RII, L-Selectin (CD62L, BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2 (CD102), IGFBP-4, Osteoprotegerin)OPG), UPAR, Activin RIB, VCAM-1 (CD106), CF, BMPR-II, IL-18 R, IL-12 R beta 1, Dtk, LBP, IGFBP-1, II-1 R rp2, IGFBP-5, IGFBP-6, MMP-1, CF, MMP-2, CF, MMP-2 (NSA-expressed), CF, MMP-7, MMp-8, MMP-10, CF, MMP-9<CF, TIMP-1, CF, TIMP-2, CF, SDF-1 alpha (PBSF)/CXCL12 (synthetic), E-Selectin (CD62E), L-Selectin (CD62L), P-Selectin (CD62P), ICAM-1 (CD54), VCAM-1 (CD106), and CD31 (PECAM-1).  
     
     
         41 . The method of  claim 39 , wherein said factors involved in cell-cell interactions are selected from the group consisting of ADAM 1, 2, 3A, 3B, 4-31, TS1-9.  
     
     
         42 . The method of  claim 39 , wherein said adhesion molecules are selected from the group consisting of Ig superfamily CAM's, Integrins, Cadherins and Selectins.  
     
     
         43 . The method of  claim 39 , wherein said extracellular matrix components are selected from the group consisting of Keratin Sulphate Proteoglycan, Laminin, Chondroitin Sulphate A, Thrombospondin-1, Heparan Sulphate, Aggregan, Biglycan, Poly-L-Ornithine, Collagen I, Collagen II, Collagen IV, Poly-D-Lysine, Ecistatin (Viper Venom), Flavoridin (Viper Venom), Kistrin (Viper Venom), Vitronectin, Superfibronectin, Fibronectin Adhesion-Promoting peptide, Fibronectin Fragment III-C, Fibronectin Fragment-30 KDA, Fibronectin-Like Polymer, Fibronectin Fragment 45 KDA, Fibronectin Fragment 70 KDA, Prostaglandin F 2 , Somatostatin, Thyrotropin Releasing Hormone, L-Thyroxine, 3,3,5-Triiodo-L-Thyronine, L-Ascorbic Acid, Asialoganglioside-GM, Disialoganglioside-GOLA, Monosialo Ganglioside-GM 1 , Monosialoganglioside-GM 2 , Monosialoganglioside-GM 3 , Lipids, Transferrin, B-Cyclodextrin, Ascorbate, Fetuin, Heparin, 2-Mercaptoethanol, Horse Serum, DMSO, Chicken Serum, Goat Serum, Rabbit Serum, Human Serum, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B, Pituitary Extract, Stromal Cell Factor, Conditioned Medium, Hybridoma Medium, d-Aldosterone, Dexamethasone, DHT, B-Estradiol, Glucagon, Insulin, Progesterone, Prostaglandin-D 2 , Prostaglandin-E1, Prostaglandin-E 2 , Prostaglandin-F 2 , Serum-Free Medium, Gene Therapy Medium, MDBK-GM Medium, QBSF-S1, Endothelial Medium, Keratinocyte Medium, Melanocyte Medium, Interleukin-10, Epidermal Growth Factor, Heparin Binding Epidermal Growth Factor, bFGF, Gly-His-Lys, Insulin Like Growth Factor-II, IGBFBP/IGF-1 Complex, C10, Cytokine Induced Neutrophil Chemoattractant 2, Cytokine Induced Neutrophil Chemoattractant 2B, Cytokine Induced Neutrophil Chemoattractant 1, Cytokine Responsive Gene-2, Endothelial Cell Growth Supplement, Interferon A, Interferon A/D, Interferon B, Interferon Inducible Protein-10, Leptin, Methylcellulose, Keratin Sulphate Proteoglycam, Laminin and Chondroitin Sulphate A.  
     
     
         44 . The method of  claim 35 , wherein said individual transfected embryonic cells or individual groups of embryonic cells are separated into separate wells of one or more multi-well plates.  
     
     
         45 . The method of  claim 35 , wherein said promoter is specifically expressed in primary or progenitor cells selected from the group consisting of endodermal cells, mesodermal cells, ectodermal cells, brain cells, heart cells, liver cells, skin cells, pancreatic cells, blood cells, reproductive cells, nerve cells, sensory cells, vascular cells, skeletal cells, immune cells, lung cells, muscle cells, and kidney cells.  
     
     
         46 . The method of  claim 35 , wherein said reporter gene encodes a polypeptide capable of producing a detectable signal, wherein said signal is coupled to transcription from said promoter.  
     
     
         47 . The method of  claim 46 , further comprising: 
 (f) quantitatively determining the amount of detectable signal; and    (g) comparing said amount of detectable signal with the amount of signal produced by the same number of said transfected embryonic cells in the absence of any test compound.    
     
     
         48 . The method of  claim 46 , wherein said detectable signal is detectable by the naked eye or after microscopic, photographic or radiographic analysis, after contacting said exposed cells with a reagent selected from the group consisting of chromogenic substrates, dyes, sugars, antibodies, ligands and primers.  
     
     
         49 . The method of  claim 46 , wherein said polypeptide is selected from the group consisting of enhanced green fluoresence protein (EGFP), luciferase, chloramphenical acetyltransferase, β-glucuronidase, β-galactosidase, neomycin phosphotransferase, alkaline phosphatase, guanine xanthine phosphoribosyltransferase and β-lactamase.  
     
     
         50 . A method for evaluating the differentiation of a totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, or a group of such cells in response to one or more compounds, comprising: 
 (a) obtaining a library of transfected totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, each transfected with at least one reporter gene, the expression of which is linked to a promoter that is activated when the cell is induced to differentiate or partially differentiate;    (b) separating individual members of said library into one or a plurality of separate vessels which may be open or closed, which vessels may be in the same or different apparatus;    (c) exposing said separate vessels of transfected cells simultaneously or sequentially to the same one or more putative differentiation-inducing compounds; and    (d) analyzing said individual members of said library in order to detect expression of said at least one reporter gene.    
     
     
         51 . The method of  claim 50 , wherein said totipotent, nearly totipotent, or pluripotent stem cells, are selected from the group consisting of inner cell mass (ICM) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryos consisting of one to about 400 cells, embryoid body cells, morula-derived cells, embryonic pluripotent cells, and cells therefrom.  
     
     
         52 . The method of  claim 50 , wherein said totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, are selected from the group consisting of human cells, primate cells, bovine cells, porcine cells, murine cells, rat cells, sheep cells, and rabbit cells.  
     
     
         53 . The method of  claim 50 , wherein said one or more putative differentiation-inducing compounds are selected from the group consisting of growth factors, cytokines, factors involved in cell-to-cell interactions, adhesion molecules and extracellular matrix components.  
     
     
         54 . The method of  claim 53 , wherein said growth factors and cytokines are selected from the group consisting of TGF-beta 2, PDGF, Fas Ligand, FGF acidic, B7-1(CD80), B7-2(CD86), DR6, IL-13 R alpha, IL-15 R alpha, GRO beta/CXCL2 (aa 39-107), IL 1-18, II-8/CXCL8, TNF-alpha, TNF-beta, GDNF, G-CSF, GM-CSF, M-GSF, PDGF-BB, PDGF-M, PDGF-AB, IL-2 sR alpha, IL-2 sR beta, Soluble TNF RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR, beta-ECGF, FGF basic (146 aa), FGF basic (157 AA), FGF1-21, TGF-alpha, TGF-beta 1, TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF, LIF sR alpha, LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF, CNTF, Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF, Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, I-309/CCL1, GRO alpha/CXCL1, GRO beta/CXCL2, GRO gamma/CXCL3, Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, IFN-gamma, Erythropoietin, Thrombopoietin, MIF, IGF-I, IGF-II, VEGF, HGF, Oncostatin M, HRG-alpha (EGF Domain), Latent TGF-beta, TGF-beta 2, CNTF R alpha, Tie-2/Fc Chimera, BMP-4, BMPR-IA, Eotaxin/CCL11, VEGF R1 (Fit-1), PDGF sR alpha, HCC-1/CCL14, CTLA-4, MCP-4/CCL13, GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A, TGF-beta sRII, Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94), TRAIL R1 (DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP, BMP-2, HVEM/VEGF R2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3 beta/CCL19, Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17, 6Ckine/CCL21, p75 Neurotrophin R (NGF R), SMDF, Neurturin, Leptin R/Fc Chimera, MIG/CXCL9, NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1 (CT-1), GFR alpha-2, BMP-5, IL-8/CXCL8 (Endothelial Cell Derived), Tie-1, Viral CMV UL146, VEGF-D, Angiopoietin-2, Inhibin A, TRANCE/RANK L, CD6/Fc Chimera, CF, dMIP-1 delta/LKN-1/CCL15(68 aa), TRAIL R3/Fc Chimera, Soluble TNF RI, Activin RIA, EphA1, E-Cadherin, ENA-70, ENA-74, Eotaxin-3/CCL26, ALCAM, FGFR1 alpha (IIIc), Activin B, FGFT1 beta (IIIc), LIGHT, FGFR2 beta(IIIb), DNAM-1, Follistatin, GFR alpha-3, gp 130, I-TAC/CXCL11, IFN-gamma RI, IGFBP-2, IGFBP-3, Inhibin B, Prolactin CF, RANK, FGFR2 beta (IIIc), FGFR4, TrkB, GITR, MSP R, GITR Ligand, Lymphotactin/XCL1, FGFR2 alpha (IIIc), Activin AB, ICAM-3 (CD50), ICAM-1 (CD54), TNF RII, L-Selectin (CD62L, BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2 (CD102), IGFBP-4, Osteoprotegerin)OPG), uPAR, Activin RIB, VCAM-1 (CD106), CF, BMPR-II, IL-18 R, IL-12 R beta 1, Dtk, LBP, IGFBP-1, II-1 R rp2, IGFBP-5, IGFBP-6, MMP-1, CF, MMP-2, CF, MMP-2 (NSA-expressed), CF, MMP-7, MMp-8, MMP-10, CF, MMP-9<CF, TIMP-1, CF, TIMP-2, CF, SDF-1 alpha (PBSF)/CXCL12 (synthetic), E-Selectin (CD62E), L-Selectin (CD62L), P-Selectin (CD62P), ICAM-1 (CD54), VCAM-1 (CD106), CD31 (PECAM-1).  
     
     
         55 . The method of  claim 53 , wherein said factors involved in cell-cell interactions are selected from the group consisting of ADAM 1, 2, 3A, 3B, 4-31, TS1-9.  
     
     
         56 . The method of  claim 53 , wherein said adhesion molecules are selected from the group consisting of Ig superfamily CAM's, Integrins, Cadherins and Selectins.  
     
     
         57 . The method of  claim 53 , wherein said extracellular matrix components are selected from the group consisting of Keratin Sulphate Proteoglycan, Laminin, Chondroitin Sulphate A, Thrombospondin-1, Heparan Sulphate, Aggregan, Biglycan, Poly-L-Ornithine, Collagen I, Collagen II, Collagen IV, Poly-D-Lysine, Ecistatin (Viper Venom), Flavoridin (Viper Venom), Kistrin (Viper Venom), Vitronectin, Superfibronectin, Fibronectin Adhesion-Promoting peptide, Fibronectin Fragment III-C, Fibronectin Fragment-30 KDA, Fibronectin-Like Polymer, Fibronectin Fragment 45 KDA, Fibronectin Fragment 70 KDA, Prostaglandin F 2 , Somatostatin, Thyrotropin Releasing Hormone, L-Thyroxine, 3,3,5-Triiodo-L-Thyronine, L-Ascorbic Acid, Asialoganglioside-GM, Disialoganglioside-GOLA, Monosialo Ganglioside-GM 1 , Monosialoganglioside-GM 2 , Monosialoganglioside-GM 3 , Lipids, Transferrin, B-Cyclodextrin, Ascorbate, Fetuin, Heparin, 2-Mercaptoethanol, Horse Serum, DMSO, Chicken Serum, Goat Serum, Rabbit Serum, Human Serum, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B, Pituitary Extract, Stromal Cell Factor, Conditioned Medium, Hybridoma Medium, d-Aldosterone, Dexamethasone, DHT, B-Estradiol, Glucagon, Insulin, Progesterone, Prostaglandin-D 2 , Prostaglandin-E1, Prostaglandin-E 2 , Prostaglandin-F 2 , Serum-Free Medium, Gene Therapy Medium, MDBK-GM Medium, QBSF-S1, Endothelial Medium, Keratinocyte Medium, Melanocyte Medium, Interleukin-10, Epidermal Growth Factor, Heparin Binding Epidermal Growth Factor, bFGF, Gly-His-Lys, Insulin Like Growth Factor-II, IGBFBP/IGF-I Complex, C10, Cytokine Induced Neutrophil Chemoattractant 2, Cytokine Induced Neutrophil Chemoattractant 2B, Cytokine Induced Neutrophil Chemoattractant 1, Cytokine Responsive Gene-2, Endothelial Cell Growth Supplement, Interferon A, Interferon A/D, Interferon B, Interferon Inducible Protein-10, Leptin, Methylcellulose, Keratin Sulphate Proteoglycam, Laminin and Chondroitin Sulphate A.  
     
     
         58 . The method of  claim 50 , wherein said individual members of said library of transfected cells are separated into separate wells of one or more multi-well plates.  
     
     
         59 . The method of  claim 50 , wherein said promoters are specifically expressed in primary or progenitor cells selected from the group consisting of endodermal cells, mesodermal cells, ectodermal cells, brain cells, heart cells, liver cells, skin cells, pancreatic cells, blood cells, reproductive cells, nerve cells, sensory cells, vascular cells, skeletal cells, immune cells, lung cells, muscle cells, and kidney cells.  
     
     
         60 . The method of  claim 50 , wherein said totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, are primate embryonic stem cells.  
     
     
         61 . The method of  claim 60 , wherein said primate embryonic stem cells are Cyno-1 cells.  
     
     
         62 . The method of  claim 50 , wherein said totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, are murine embryonic stem cells.  
     
     
         63 . The method of  claim 62 , wherein said murine embryonic stem cells each contain a gene-trap vector insertion.  
     
     
         64 . The method of  claim 63 , wherein said murine embryonic stem cells comprise members of OmniBank® and/or ES cell mutants corresponding to a gene-trap sequence tag (GTST) reference library.  
     
     
         65 . The method of  claim 50 , further comprising: 
 isolating primary and/or progenitor cells from reference tissues and placing said primary and/or progenitor cells into separate vessels of a microarray thereby forming a control reference library; and    after exposing said separate vessels of totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, to the said one or more putative differentiation-inducing compounds either simultaneously or sequentially;    comparing said individual totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, or groups of cells, to said reference library in order to evaluate the differentiation of said individual cells or groups of cells.    
     
     
         66 . The method of  claim 65 , wherein said reference library of primary cells is constructed in one or more multi-well plates.  
     
     
         67 . The method of  claim 65 , wherein said primary and/or progenitor cells for said reference library include one or more of brain cells, heart cells, liver cells, skin cells, pancreatic cells, blood cells, reproductive cells, nerve cells, sensory cells, vascular cells, skeletal cells, immune cells, lung cells, muscle cells, and kidney cells.  
     
     
         68 . The method of  claim 65 , wherein said totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, and said reference library cells are compared using functional assays specific for the particular primary cells in the reference library.  
     
     
         69 . The method of  claim 68 , wherein said functional assays measure the production of enzymes or metabolites produced by said reference primary and/or progenitor cells.  
     
     
         70 . The method of  claim 65 , wherein said exposed totipotent, nearly totipotent, or pluripotent stem cells, or cells therefrom, and said reference library cells are compared by testing for the presence of specific cell surface markers or receptors.  
     
     
         71 . The method of  claim 70 , wherein the presence of said cell surface markers is detected using labeled antibodies or ligands specific for said cell surface markers or receptors.  
     
     
         72 . The method of  claim 1 , wherein said screening step comprises making multiple measurements over a course of time to identify agents or conditions that promote increased survival of the cells over the period of the assay.  
     
     
         73 . The method of  claim 1 , wherein said screening step comprises making multiple measurements over a course of time to identify agents or conditions that promote an increase in cell number over the period of the assay.  
     
     
         74 . A library of two or more gene trap stem cell lines used simultaneously together to screen to detect agents or conditions that affect differentiation, survival, or proliferation of the stem cells.  
     
     
         75 . The library of  claim 74 , wherein one or more of said gene trap stem cell lines is uni-nodal; i.e., contains a reporter gene that is activated in only one cell type upon differentiation or partial differentiation of the stem cells.  
     
     
         75 . The library of  claim 74 , wherein one or more of said gene trap stem cell lines is multi-nodal; i.e., contains a reporter gene is activated in two or more different cell types upon differentiation or partial differentiation of the stem cells.  
     
     
         76 . A method for inducing differentiation of a stem cell to form cells of mesodermal lineage, comprising exposing the stem cells to Flt-3.  
     
     
         77 . The method of  claim 76  wherein said cells of mesodermal lineage are vascular endothelial cells.  
     
     
         78 . A method for inducing differentiation of a stem cell to form cells of mesodermal and neural lineage, comprising exposing the stem cells to TGF-beta-1.  
     
     
         79 . A method for inducing differentiation of a stem cell to form cells selected from the group consisting of cells of endothelial lineage, and cells of endodermal lineage or appearance, comprising exposing the stem cells to tenascin.  
     
     
         80 . A method for inducing differentiation of a stem cell comprising exposing the stem cells to Tie-1.  
     
     
         81 . The method of  claim 80 , further comprising exposing the stem cells to a Fc moiety.  
     
     
         82 . A method for inducing differentiation of a stem cell to form fibroblasts and/or other cells of connective tissue comprising exposing the stem cells to BMP-2.  
     
     
         83 . A method for inducing differentiation of a stem cell to form myocardial cells comprising exposing the stem cells to endothelial inducer cells.  
     
     
         84 . A method for inducing differentiation of a stem cell to form cells of mesodermal lineage comprising exposing the stem cells to fibroblast inducer cells.  
     
     
         84 . The method of  claim 84 , further comprising exposing the stem cells to FGF-4 and TGF-beta-1.